Live attenuated Leishmania vaccines

ABSTRACT

Targeted disruption of the centrin gene leads to attenuation of growth in the  Leishmania . Preferred embodiments of the invention provide attenuated strains of  Leishmania  useful for the preparation of immunogenic preparations including vaccines against a disease caused by infection with a virulent  Leishmania  strain and as tools for the generation of immunological and diagnostic reagents. Other preferred embodiments provide related immunogenic compositions, methods of generating an immune response, methods for producing a vaccine, and methods of forming attenuated strains of  Leishmania.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 11/364,682, filed Feb. 28, 2006, which is acontinuation of International Patent Application No. PCT/US2004/028008,filed Aug. 27, 2004, designating the U.S. and published in English onMar. 10, 2005 as WO 2005/021030, which claims the benefit of U.S.Provisional Application No. 60/498,816, filed Aug. 29, 2003, and U.S.Provisional Application No. 60/549,507, filed Mar. 1, 2004, all of whichare hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

Targeted disruption of the centrin gene leads to attenuation of growthin Leishmania.

BACKGROUND OF THE INVENTION

Vector borne disease, Leishmaniasis currently threatens 1.5-2.0 millionpeople annually with an estimated death toll of 50,000 persons/year in88 countries around the world (Ganguly, N. K. 2002 Special Program forResearch and Training in Tropical Diseases News. Geneva: United NationsDevelopment Programme/World Bank/World Health Organizations, Report no.68; Singh, N. et al. 2003 J Infect Dis 188: 600-607). The parasiticdisease has a wide range of clinical symptoms, which include thecutaneous form caused by Leishmania aethiopica, L. mexicana, L. majorand L. tropica, the mucocutaneous form by L. braziliensis and thevisceral form by L. chagasi, L. donovani and L. infantum, which is fatalif not treated in the case of L. donovani. Vector control is difficultand complicated by the diverse ecology of many species of sand flyvectors and animal reservoirs (Sharifi, I. et al. 1998 Lancet351:1540-1543). Treatment for these diseases involves chemotherapy usingantimony-based drugs, which is less effective in immunocompromisedindividuals (Handman, E. 2001 Clin Microbiol Rev 14:229-243). Noeffective vaccine is yet available for any of these diseases.

SEGUE TO THE INVENTION

The parasite has a digenic life style with one form called thepromastigote that resides extracellularly in the mid-gut of the sand-flyvector and the other the amastigote form that multipliesintra-cellularly in the vertebrate macrophages. These two forms havebeen adapted to grow under in vitro conditions (Debrabant, A. et al.2003 Int J Parasitol 33:257-267; Goyard, S. et al. 2003 Mol BiochemParasitol 130:31-42; Joshi, M. et al. 1993 Mol Biochem Parasitol58:345-354). Defects in growth have been correlated with the attenuationof virulence in Leishmania (Denise, H. et al. 2003 Infect Immun71:3190-3195; Handman, E. 2001 Clin Microbiol Rev 14:229-243). In orderto develop an avirulent Leishmania deficient for growth in vertebratemacrophages we generated a gene-targeted deletion for a key growthregulating gene, L. donovani centrin (LdCEN) (Selvapandiyan, A. et al.2001 J Biol Chem 276:43253-43261). The centrins are basal bodyassociated calcium binding proteins. Centrin has been characterized inother eukaryotes from unicellular Chlamydomonas (Koblenz, B. et al. 2003J Cell Sci 116:2635-2646; Salisbury, J. L. 1995 Curr Opin Cell Biol7:39-45) to humans (Errabolu, R. et al. 1994 J Cell Sci 107:9-16; Gavet,O. et al. 2003 Mol Biol Cell 14:1818-1834; Matei, E. et al. 2003Biochemistry 42:1439-1450; Salisbury, J. L. et al. 2002 Curr Biol, 12:1287-1292). Fibers composed of the centrin-binding protein Sfi1p andcentrin act as contractile or elastic connections betweencentrioles/basal bodies and other elements of the centrosome to mediatedynamic changes in its overall structure (Kilmartin, J. V. 2003 J CellBiol 162:1211-1221; Salisbury, J. L. 2004 Curr Biol 14:R27-29). Proteinsequence similarity and immunoreactivity confirmed that Leishmaniacentrin is a homolog of human centrin 2 (Selvapandiyan, A. et al. 2001 JBiol. Chem. 276:43253-43261). LdCen-p protein is highly expressed inrapidly growing parasites of both forms and declines when cells reachstationary phase (Selvapandiyan, A. et al. 2001 J Biol. Chem.276:43253-43261). Expression of N-terminal deleted centrin in theparasite significantly reduces the growth rate of both the stages of theparasite (Selvapandiyan, A. et al. 2001 J Biol Chem. 276:43253-43261).These studies indicate that centrin has an important functional role inLeishmania growth. Many earlier studies have also elucidated theimportance of the centrin gene in various cell processes, for example,conditional mutations in yeast centrin (CDC31) resulted in failure ofduplication of the spindle pole body (yeast centrosome) and arrest inmitosis (Baum, P. et al. 1986 PNAS USA. 83:5512-5516; Paoletti, A. 2003Mol Biol Cell 14:2793-2808). Silencing the centrin gene in the waterfern Marsilea vestita effectively inhibits the formation of motile cells(Klink, V. P. and Wolniak, S. M. 2001 Mol Biol Cell 12:761-776).Silencing of human centrin 2 in HeLa cells impaired centrioleduplication and result in failure of cytokinesis (Salisbury, J. L. etal. 2002 Curr Biol. 12: 1287-1292). A centrin deficient mutant ofChlamydomonas was non-flagellate due to defects in the flagellar rootsystem (Koblenz, B. et al. 2003 J Cell Sci 116:2635-2646). Abnormalcentrosomes were noticed in human tumor cells due to the presence ofexcess levels of many centrosome proteins including centrin (Lingle, W.L. et al. 1998 PNAS USA 95:2950-2955; Lingle, W. L. and Salisbury, J. L.1999 Am J Pathol 155:1941-1951). In order to further analyze thecharacteristics of centrin in the important human parasite, L. donovani,we targeted LdCEN gene for a null mutation.

Various types of Leishmania vaccine strategies have been attempted withlittle success. Attenuated parasitic strains obtained throughγ-irradiation, long-term in vitro culture, selection for temperaturesensitivity, or chemical mutagenesis, possess genetically undefinedmutations (Handman, E. 2001 Clin Microbiol Rev 14:229-243). Suchparasites can revert back to the virulent form or persist for longperiods of time. Persistence of asymptomatic Leishmania infectionsraises the risk of subsequent reactivation, especially in AIDS whereleishmaniasis is an opportunistic infection (Titus, R. G. et al. 1995PNAS USA 92:10267-10271). An alternate promising strategy is to generatenonreversible genetically defined attenuated parasites by deleting knownessential genes. To date, few genetically defined mutations have beencarried out in Leishmania. Dihydrofolate reductase-thymidylate synthaseand Leishmanolysin genes were deleted in L. major (Cruz, A. et al. 1991PNAS USA 88:7170-7174; Joshi, P. B. et al. 2002 Mol Biochem Parasitol120:33-40; Joshi, P. B. et al. 1998 Mol Microbiol 27:519-530; Titus, R.G. et al. 1995 PNAS USA 92:10267-10271) and genes involved inLipophosphoglycan were deleted in L. mexicana and L. major (Ilg, T. 2000Embo J 19:1953-1962; Spath, G. F. et al. 2000 PNAS USA 97:9258-9263;Spath, G. F. et al. 2003 PNAS USA 100:9536-41). While the outcome ofvaccine efficacy testing of these mutant L. major/mexicana strains isbeing evaluated, we envision the generation of a nonreversiblegenetically defined attenuated vaccine for L. donovani based on theoutcome of the studies on centrin gene deletion.

Studying the characteristics of genes and generating mutant organismsthrough silencing the corresponding mRNA to translate into proteins, viaRNA interference (RNAi) approach, is successful in many eukaryotesincluding the parasites T. brucei (Wang, Z. et al. 2000 J Biol Chem 275:40174-40179) and Plasmodium falciparum (Malhotra, P. et al. 2002 MolMicrobiol 45:1245-1254). This approach has so far been unsuccessful inLeishmania probably due to lack of RNAi processing machinery in theseorganisms (Robinson, K. A. and Beverley, S. M. 2003 Mol BiochemParasitol 128:217-228). However, gene replacement through homologousrecombination is still a powerful method for altering and testing genefunction (Capecchi, M. R. 1989 Science 244:1288-1292; Cruz, A. et al.1991 PNAS USA 88:7170-7174). Unlike many other eukaryotes, Leishmania isdiploid throughout its life cycle. Hence it may be necessary to deleteboth the alleles of a gene such as by targeting with two differentmarker genes (Cruz, A. et al. 1991 PNAS USA 88:7170-7174). In thisdisclosure we describe a stepwise disruption of the two alleles ofLdCEN, using genes resistant to antibiotics hygromycin B and G418(Geneticin) and characterization of the LdCEN null mutant parasites fortheir growth both extracellularly as promastigotes as well as axenicamastigotes and inside macrophages. The purpose of the disclosure is toestablish correlation between centrin expression and parasite growth andcharacterize the LdCEN null mutants for use as a live attenuatedvaccine.

SUMMARY OF THE INVENTION

Targeted disruption of the centrin gene leads to attenuation of growthin Leishmania. Preferred embodiments of the invention provide attenuatedstrains of Leishmania useful for the preparation of immunogenicpreparations including vaccines against a disease caused by infectionwith a virulent Leishmania strain and as tools for the generation ofimmunological and diagnostic reagents. Other preferred embodimentsprovide related immunogenic compositions, methods of generating animmune response, methods for producing a vaccine, and methods of formingattenuated strains of Leishmania.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic diagram showing design and use of constructs for LdCENgene disruption in the L. donovani genome. Constructs 1 and 2 show hygor neo genes respectively, flanked on the 5′ and 3′ sides with LdCEN5′UTR and 3′UTR respectively. The lengths of the antibiotic resistantgenes and the LdCEN untranslated regions have been described inExample 1. M-seq: 14 nucleotides upstream and 12 nucleotides downstreamof the ATG start codon of 3′NT/Nu gene. BamHI, KpnI, SalI and SpeI arethe restriction sites.

FIG. 2 (A) Schematic drawing of the centrin locus in L. donovani withSalI restriction sites. Disrupting the centrin gene with either hyg orneo gene generates an additional SalI site at the 5′ end of theselectable marker genes. (B) Southern blot analysis of the genomic DNAof Leishmania wild type (+/+), LdCEN single (+/−) and double (−/−)allele disrupted parasites with hyg, neo and LdCEN genes as ³²P labeledprobes. The genomic DNA from the parasites was cut with the restrictionenzyme SalI and used in the analysis. (C) Western blot analysis of thelysates of +/+, +/− and −/− parasites using αLdCen Ab (Selvapandiyan, A.et al. 2001 J Biol Chem, 276:43253-43261). Lower panel: After theprotein transfer the membrane was stained with Ponceau S to control forprotein loading.

FIG. 3 The effect of LdCEN disruption on the growth of in vitro grownLeishmania promastigotes (A) and axenic amastigotes (B) of wild type(+/+), centrin single allele disrupted (+/−) and centrin both thealleles disrupted (−/−). The cells were grown in the absence ofantibiotics. Initial cell density in the culture was 0.1×10⁷ cells/ml.Data represent the mean±SD of four independent experiments.

FIG. 4 Indirect immunofluorescence analysis of centrin distribution in(A) wild type promastigote and (B) amastigote, and (C) LdCEN^(−/−)promastigote and (D) amastigote cells. Centrin staining at the flagellarbase and along the axoneme is evident in promastigote cells and at theflagellar basal apparatus in wild type amastigote cells. LdCEN^(−/−)amastigote cells are largely devoid of specific staining. Bar=5 μm.

FIG. 5 Electron microscopy of the flagellar apparatus of (A) wild typepromastigote and (B) amastigote, and (C) LdCEN^(−/−) promastigote and(D) amastigote cells. Evidence of duplication with more than two basalbodies was frequently observed in (E) wild type promastigote and (F)amastigote, and (G) LdCEN^(−/−) promastigote cells, but not in (H)amastigote cells where more than two basal bodies were never seen.Bar=0.5 μm.

FIG. 6 Flow cytometric analysis of the DNA content of L. donovani wildtype (+/+) and centrin deficient (−/−) parasites. Promastigotesinoculated at 1×10⁶ cells/ml in axenic amastigote medium were allowed togrow 24 or 48 hr. The resulting axenic amastigotes were harvested,fixed, stained with propidium iodide and subjected tofluorescence-activated cell scan analysis. The percent of cells in eachcell cycle stage, G1, S or G2/M, is plotted for the 24 hr time point(bar graph to the left) and 48 hr time point (bar graph to the right).Solid bars indicate the wild type cells (+/+) and hatched bars indicatethe centrin deficient (−/−) cells. Data represent the mean±SD of threeindependent experiments. *P<0.008 and **P<0.0005 student's t-test.

FIG. 7 (A) Phase contrast images of L. donovani wild type (+/+) andcentrin deficient (−/−) axenic amastigotes after 48 hr of in vitroculture. (B) Para-formaldehyde fixed +/+ and −/− axenic amastigote cellsfrom 48 hr culture were stained with DAPI and viewed under a fluorescentmicroscope. Right panel: pale blue (light gray) spots are kinetoplasts,larger dark blue (dark grey) regions are nuclei. Left panel: phasecontrast image of the same field shown in the right panel (C)Transmission Electron Micrograph of Leishmania wild type (+/+) (1) andmutant (−/−) (2 and 3) axenic amastigotes after 48 hr in culture. Shownare one representative field for wild type (+/+) and two representativefields for centrin knockout mutants (−/−). K—kinetoplast, M—microtubulesand N—Nucleus are indicated. All images original magnification: 400×.(D) Quantitative analysis of the number of DAPI stained kinetoplasts inthe viable LdCEN^(−/−) axenic amastigotes with increasing time inculture. Cells, with one, two or more than four kinetoplasts, werecounted and individually plotted as percent of cells showing each numberof kinetoplasts/cell. The results were obtained from at least 150 viablecells (cells not staining to tryphan blue) observed in each case. Datarepresent the mean±SD of three independent experiments.

FIG. 8 Percent of cells positive for the uptake of PI. FACS analysis ofparasites of the +/+ (solid bars) and −/− (hatched bars) genotypessampled over time after inoculation into axenic amastigote culturemedium. Fluorescing cell populations were measured for PI at FL-3channel. For each sample 10,000 fluorescent events were measured.

FIG. 9 (A) Percent of cells positive for PPL cleavage activity. FACSanalysis of parasites analyzed in the above experiment (FIG. 6) weremeasured simultaneously for the PPL cleavage activity at FL-1 channel.(B) Percent of TUNEL positive +/+ and −/− axenic amastigotes at 0, 24and 36 hr incubation periods. The culture at 0 hr time point indeed werethe exponentially growing promastigote parasites used to initiate theaxenic amastigote culture for the assay. At each time point at least 200total cells were counted. Data represent the mean±SD of threeindependent experiments. *P<0.008 student's t-test. (C) TUNEL assayimages of samples included in the cell counts in part E, observed underthe fluorescence microscope. DAPI images false colored red (dark grey)show the staining of both the kinetoplasts and the nuclei (images 1 and2). The TUNEL positive nuclei and kinetoplasts show green fluorescence(light grey) (images 3 and 4). DAPI and TUNEL images were merged andshown (images 5 and 6). All images original magnification: 1000×.

FIG. 10 (A) Percent infected macrophages (top panel), number ofparasites per 100 total macrophages (middle panel) and number ofparasites per infected macrophage (bottom panel) at variouspost-infection time points. Results from three independent experimentswere averaged and plotted with error bars indicating the standarddeviation. *P<0.005 and **P<0.0001 student's t-test. (B) Lightmicroscopy of the human macrophages infected with the +/+ and −/−parasites incubated for 120 and 240 hr. Am—amastigotes; M—multinucleatedparasites; P—phagolysosome in a macrophage. All images originalmagnification: 400×.

FIG. 11 (A) Western blot analysis on the cell lysate from axenicamastigotes of Leishmania wild type (+/+), LdCEN knockout (−/−), LdCENknockout parasites with centrin added back by transfection of a centrinexpression plasmid (pXG-PHLEO-LdCEN) (−/−AB) and wild type similarlyover expressing centrin (+/+AB). Blots were developed using αLdCen Ab.(B) Growth analysis of the axenic amastigote parasites of the +/+, −/−,+/+AB and −/−AB. Data represent the mean±SD of three independentexperiments.

FIG. 12 A, B Centrin sequence from different species of Leishmania.Nucleotide sequence for centrin isolated from Leishmania donovani,Leishmania infantum, Leishmania amazonensis, Leishmania major,Leishmania mexicana, and Leishmania tropica.

BRIEF DESCRIPTION OF THE SEQUENCES

Sequence SEQ ID NO. Leishmania donovani centrin SEQ ID NO: 1 Leishmaniainfantum centrin SEQ ID NO: 2 Leishmania amazonensis centrin SEQ ID NO:3 Leishmania major centrin SEQ ID NO: 4 Leishmania mexicana centrin SEQID NO: 5 Leishmania tropica centrin SEQ ID NO: 6

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Recently, we identified and cloned multiple centrin genes from L.donovani. Homologues of centrin have been shown to be involved induplication and segregation of centrosomes in higher eukaryotes.Expression of Leishmania centrin correlates with the growth phase of theparasite. In order to understand the role of centrin in the parasitegrowth, we created Leishmania deficient in one of its centrin genes(LdCEN) by double homologous gene replacement. We found that centrinnull mutants (LdCEN^(−/−)) had a selective growth arrest as axenicamastigotes and not as promastigotes. The axenic amastigotes showedfailure of basal body duplication and failure of cytokinesis resultingin multinucleated ‘large’ cells. Flowcytometry analysis confirmed thatthe mutant parasites have a cell cycle arrest at the G2/M stage.Increased caspase like activity and concomitant TUNEL positivity wasobserved in centrin mutant amastigotes compared to wild type cellsindicating the activation of programmed cell death pathway, whichsubsequently could result in loss of viable cells. When LdCEN^(−/−)promastigotes were used to infect macrophage cells in culture, growth ofthe intracellular amastigotes was inhibited and large multinucleatedparasite cells similar to those observed in vitro resulted. Uponre-expression of recombinant centrin in LdCEN^(−/−) parasites, growthwas restored in the mutant parasites similar to wild type parasites.These results demonstrate the direct involvement of centrin expressionin Leishmania growth. Further, this is the first report where targeteddisruption of a gene involved in cytokinesis leads to stage specificattenuation in a parasite. Leishmania centrin null mutants with a growthdefect in axenic amastigotes are envisioned as being useful as anattenuated parasite vaccine against leishmaniasis.

Further Description Section I

The present invention is directed towards the provision of attenuatedstrains of Leishmania. The attenuated strains are useful for thepreparation of immunogenic preparations including vaccines againstdisease caused by infection by a virulent Leishmania strain and as toolsfor the generation of immunological and diagnostic reagents.

In accordance with one aspect of the present invention, there isprovided an attenuated strain of Leishmania wherein at least one gene ofthe strain contributing to virulence thereof and expressed in both thepromastigote and amastigote stages of the life cycle of the strain hasbeen functionally disabled by, for example, a deletion of at least aportion of the gene or by mutagenesis.

In another aspect of the invention, there is provided an attenuatedstrain of Leishmania wherein both wild-type copies of a gene of thestrain contributing to virulence thereof have been functionallydisabled. The gene contributing to the virulence of the strain in thisaspect of the invention may be one expressed in both the promastigoteand amastigote stages of the life cycle of the strain.

The gene may contribute to the ability of the strain to infect orsurvive within macrophages and, in a particular embodiment, may encodeLeishmania centrin. The attenuated Leishmania strain may be selectedfrom the group consisting of Leishmania donovani, Leishmania infantum,Leishmania chagasi, Leishmania major, Leishmania tropica, Leishmaniaaethiopica, Leishmania braziliensis, Leishmania mexicana, and Leishmaniaamazonensis.

The six species of Leishmania recognized to cause disease in humans(Table 1) are very similar morphologically but produce strikinglydifferent pathological responses. The only feature common to all is thechronicity of disease manifestations. The infection may be predominantlyvisceral, as in visceral leishmaniasis or Indian kala-azar, orrestricted to the skin, as with the chronic ulcer of Oriental sore, orspreading to the mucous membranes to produce the disfiguring SouthAmerican espundia (Handman, E. et al. 2001 Clinical Microbiology Review14:229-243.)

TABLE 1 Leishmania species pathogenic for humans, their vectors, hostrange and disease manifestations Species Host range Main vector Diseasemanifestations L. donovani (India and Africa) Dogs, savannah rodents, P.argentipes, Visceral leishmaniasis (kala and related L. infantum humansL. longipalpis azar), post-kala-azar dermal (Mediterranean region, theleishmaniasis (PKDL) Middle East, and Asia) and L. chagasi (SouthAmerica) L. major Desert and savannah P. papatasi Cutaneousleishmaniasis rodents; Rhombomys, (rural, wet Oriental sore) Psammomys,Arvicanthis L. tropica Humans P. sergenti Cutaneous leishmaniasis(urban, dry Oriental sore), visceral leishmaniasis L. aethiopica Rockhyrax P. longipes Cutaneous leishmaniasis, diffuse cutaneousleishmaniasis L. braziliensis complex Sloth, dog L. umbratilis andCutaneous leishmaniasis, many others mucocutaneous leishmaniasis L.mexicana complex, e.g., Forest rodents L. flaviscutellata, Cutaneousleishmaniasis, L. mexicana amazonensis L. olmeca diffuse cutaneousleishmaniasis

The sequence of Leishmania donovani centrin (centrin that has beendeleted in L. donovani) is provided in GenBank accession number AF406767and FIG. 12.

The sequences of centrins identical to L. donivani centrin found inother species are: Leishmania infantum centrin, Leishmania amazonensiscentrin, Leishmania major centrin, Leishmania mexicana centrin, andLeishmania tropica centrin, all provided in FIG. 12.

In a further aspect, the present invention provides an immunogeniccomposition comprising the attenuated strains as provided herein. Theimmunogenic composition may be formulated as a vaccine for in vivoadministration to a host, such as a primate including humans, to conferprotection against disease caused by a virulent strain of Leishmania,including Leishmania donovani, Leishmania infantum, Leishmania chagasi,Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmaniabraziliensis, Leishmania mexicana, and Leishmania amazonensis.

In an additional aspect, the invention provides a method of generatingan immune response in a host, such as a primate including humans,comprising administering thereto an immunoeffective amount of theimmunogenic composition, as provided herein. In a particular aspect, theimmunogenic composition may be formulated as a vaccine for in vivoadministration to the host to confer protection against disease causedby a virulent strain of Leishmania, including Leishmania donovani,Leishmania infantum, Leishmania chagasi, Leishmania major, Leishmaniatropica, Leishmania aethiopica, Leishmania braziliensis, Leishmaniamexicana, and Leishmania amazonensis.

In yet an additional aspect, there is provided a method for producing avaccine for protection against a disease caused by infection by avirulent strain of Leishmania, including Leishmania donovani, Leishmaniainfantum, Leishmania chagasi, Leishmania major, Leishmania tropica,Leishmania aethiopica, Leishmania braziliensis, Leishmania mexicana, andLeishmania amazonensis, and comprising administering the immunogeniccomposition as provided herein to a test host to determine an amount andfrequency of administration thereof to confer protection against diseasecaused by infection by the Leishmania parasite and formulating theimmunogenic composition in a form suitable for administration to atreated host, including humans, in accordance with said determinedamount and frequency of administration.

In a further aspect of the invention, there is provided a method offorming an attenuated strain of Leishmania that comprises identifying agene of a Leishmania strain contributing to the virulence thereof andexpressed in both the promastigote and amastigote stages of the lifecycle of the strain, and functionally disabling the gene.

These virulence genes may be functionally disabled by, for example,deletion or mutation, including insertional mutagenesis and,furthermore, the wild-type Leishmania gene may be replaced by thefunctionally disabled gene. The virulence genes may be functionallydisabled by, for example, replacing the gene by a selectable antibioticresistance gene by homologous recombination following transformation ofthe Leishmania organism with a fragment of DNA containing the antibioticresistance gene flanked by 5′- and 3′-non-coding DNA sequences.

This method can be used to generate the attenuated variants ofLeishmania and the residual pathogenicity of the attenuated variants canbe assessed in mice and hamsters. Deletion of the genes that areselectively expressed results in an attenuated strain that cannotsurvive in humans but generates a protective immune response. Attenuatedstrains of Leishmania as provided herein would be useful as livevaccines against the diseases caused by Leishmania.

Advantages of the present invention include the provision of safe andattenuated strains of Leishmania for the preparation of immunogeniccompositions including vaccines and for the generation of immunologicaland diagnostic reagents.

Further Description Section II

It is among the objects of the present invention to provide an improvedLeishmania vaccine. More particularly it is a preferred object of thepresent invention to provide a live attenuated Leishmania vaccine.

In one aspect the present invention provides the use of a mutantLeishmania in the preparation of a vaccine, wherein the mutantLeishmania comprises a defective centrin gene, such that the mutantLeishmania is substantially incapable of expressing a functionallyactive form of centrin protein encoded by said gene

A further aspect of the invention relates to the vaccine itself.

While the present description refers mainly to the use of promastigotesin the preparation of a vaccine, it is to be understood that pureamastigotes or amastigotes in mammalian cells may be used asalternatives.

The mutant Leishmania may be selected from all species of Leishmaniaincluding Leishmania donovani, Leishmania infantum, Leishmania chagasi,Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmaniabraziliensis, Leishmania mexicana, and Leishmania amazonensis.

A “defective centrin gene” is one that is substantially incapable ofencoding for a native centrin protein or a functional equivalentthereof. Thus, a “defective centrin gene” means that the centrin genehas been modified by a deletion, insertion, substitution (or otherchange in the DNA sequence such as rearrangement) such that the centringene is generally incapable of expressing a functionally competentcentrin protein from said gene. It will be appreciated that modificationmay also extend to the regulatory regions of the gene, providing thatthe result is that a functionally competent centrin protein encoded bythe particular gene is not expressed.

The “defective centrin gene” may however be capable of expressing adefective centrin protein that is functionally inactive. Such adefective centrin protein may however be antigenic or immunogenic, suchthat a host may elicit an immune response to the defective centrinprotein.

If the mutant is for example a Leishmania donovani mutant then saidcentrin gene is a single copy gene encoding said centrin protein.

The present inventors have now also identified or may identify thecorresponding centrin gene in Leishmania donovani, Leishmania infantum,Leishmania chagasi, Leishmania major, Leishmania tropica, Leishmaniaaethiopica, Leishmania braziliensis, Leishmania mexicana, and Leishmaniaamazonensis, such that Leishmania donovani, Leishmania infantum,Leishmania chagasi, Leishmania major, Leishmania tropica, Leishmaniaaethiopica, Leishmania braziliensis, Leishmania mexicana, and Leishmaniaamazonensis centrin mutants may also be produced as described herein andused in the preparation of a vaccine.

Thus, in a further aspect the present invention provides a vaccineformulation comprising a mutant Leishmania donovani, Leishmaniainfantum, Leishmania chagasi, Leishmania major, Leishmania tropica,Leishmania aethiopica, Leishmania braziliensis, Leishmania mexicana, orLeishmania amazonensis wherein a centrin gene has been made defective asdescribed herein.

Centrin genes of the present invention that are subsequently incapableof expressing a functionally competent centrin protein may be rendereddysfunctional by any one or more ways for example:

(i) A deletion of the entire centrin protein coding region of thecentrin gene from a wild type Leishmania genome. The deletion should besuch so as not to substantially affect the expression of other geneproducts from the Leishmania parasite genome.

(ii) A deletion of a portion of the centrin protein coding region from awild type Leishmania genome. A “portion of the centrin protein codingregion” means a polynucleotide fragment that by its deletion from thecentrin protein coding region is sufficient to render any centrinprotein or fragment or fragments thereof encoded and/or expressiblethereby, substantially incapable of a physiological activityattributable to that of a functional centrin protein produced by a wildtype parasite. The deleted portion of the centrin gene may be composedof a deletion of a small number of nucleotides, for example, 1, 2 ormore nucleotides. Such deletions within the centrin gene can be achievedusing recombinant DNA technology. Thus, the translational open readingframe (ORF) for a centrin gene can be altered resulting in theproduction of a protein that lacks the physiological functionality orfunctional competence of a centrin protein derived from wild typeLeishmania. The skilled addressee will also appreciate that suchdeletions in the translational ORF of the centrin gene may also giverise to a dysfunctional gene that is substantially incapable of codingfor a functionally competent centrin protein, truncated centrin proteinor polypeptide fragment thereof. Such proteins/polypeptides, ifproduced, generally lack the functional competence typical of the fulllength centrin protein.

(iii) The deletion of the or a portion of the centrin gene as describedin (i) or (ii) above will leave a “gap” in the centrin gene. A suitablepolynucleotide such as a gene or gene fragment thereof may be insertedinto the “gap”. Gene insertions can include genes that expresspolypeptides capable of augmenting an immune response, such as mammaliancytokines, for example, γ interferon or other genes such as markergenes. Suitable marker genes may include but are not restricted to genesencoding enzymes, for example thymidine kinase, or genes encodingantibiotic resistance to such as, puromycin, tunicamycin, hygromycin,neomycin, phleomycin, nourseothricin and the like. Generally thesegenes, if any, may be employed in a centrin gene deletion. Mutants ofthe invention should be such so as not to cause substantial deleteriousor long lasting side-effects to a recipient animal.

It is typical to utilize a system that generates drug resistancemarker-exploiting mutants. Such a system may involve sequential roundsof targeted gene disruption using positive or negative selection. In thegeneration of centrin protein double allele null mutants, typically,each of said two single copy centrin genes will be targeted fordisruption independently and subsequently multiple mutants will begenerated. The hygromycin (hyg) gene may be used as a positiveselectable marker for the antibiotic hygromycin. The viral HSV thymidinekinase gene (tk) may be used as a negative selectable marker inconjunction with the drug ganciclovir (Lebowitz, J. H. 1994 Methods CellBiol. 45:65-78).

For example, wild type Leishmania may be transfected with a constructcontaining both the hyg and tk genes arranged in tandem in order todelete one allele of a centrin gene. Selection with hygromycin willallow transformants to be selected in which the particular centrin genehas been deleted as, for example, described previously (Mottram et al.1996 PNAS USA. 93:6008-13) A second round of transfection would then beperformed with a “null targeting fragment” containing centrin geneflanking DNA that will delete the hyg/tk DNA that has been integratedinto the centrin locus. Cells in which the tk gene remains may then bekilled by the ganciclovir drug, whereas mutants in which the centringene and the drug markers have been deleted will grow. In this mannerone allele of the centrin gene will have been deleted and no exogenousDNA will remain in the centrin locus. The procedure will then berepeated for the second centrin allele as Leishmania is diploid, toproduce a centrin gene null mutant. The entire procedure may then berepeated if necessary in order to produce a viable centrin gene doublenull mutant.

In a preferment there is provided a Leishmania centrin gene double nullmutant comprising deletions in said centrin regions within theLeishmania genome. The deletion should be such that coding sequences forother gene products of the Leishmania, upstream and/or downstream fromthe centrin domains, are not substantially affected. That is to say thatother gene products ordinarily having an immunogenic function and thatare expressed in Leishmania substantially retain their immunogenicfunction.

The deletion generally has to be made in said centrin genes in positionssuch that any mutant Leishmania within a host cell retains a sufficientfunction to elicit an immune response in a host animal, such as a dog orhuman. If the prophylactic and/or therapeutic effect of an appropriateLeishmania mutant of the present invention is to be augmented, anappropriate adjuvant, such as a cytokine, for example, γ interferon(γ-IFN) can also be employed as a component of a vaccine orpharmaceutical composition of the invention.

Optionally, such centrin gene null Leishmania mutants may be furthermodified to express a dysfunctional form of a centrin protein, such asthe centrin protein that is not expressed by the mutant. Thus, themutant may express a centrin protein that is functionally inactive, butthat is antigenic or immunogenic. For example, to produce an inactivecentrin protein the active site can be changed by site-directedmutagenesis. This mutation should result in the production of fulllength centrin protein that is functionally inactive. The gene encodingthe inactive centrin protein can then be re-introduced into a centringene null mutant by homologous recombination using unique sequence thatflank the centrin gene.

In a further embodiment of the invention there is provided a host cellcomprising a centrin gene null Leishmania mutant of the presentinvention. The host cell may for example be a macrophage or similar celltype known to those skilled in art.

Centrin gene null Leishmania mutants of the present invention may beapplied directly to the cells of an animal in vivo, or by in vitroinfection of cells taken from the said animal, which cells are thenintroduced back into the animal. Leishmania centrin gene null mutantsmay be delivered to various tissues of the animal body including muscle,skin or blood cells thereof. The Leishmania centrin gene null mutant maybe injected into for example, muscle or skin using a suitable syringe.

Centrin gene null Leishmania mutants for injection may be prepared inunit dosage form in ampoules, or in multidose containers. The parasitesmay be present in such forms as suspensions, solutions, or emulsions inoily or preferably aqueous vehicles. For any parenteral use,particularly if the formulation is to be administered intravenously, thetotal concentration of solutes should be controlled to make thepreparation isotonic, hypotonic, or weakly hypertonic. Nonionicmaterials, such as sugars, are preferred. Any of these forms may furthercomprise suitable formulatory agents, such as starch or sugar, glycerolor saline. The compositions per unit dosage, whether liquid or solid,may contain from 0.1% to 99% of parasite material.

In a further embodiment of the invention there is provided a vaccineagainst Leishmania comprising a centrin gene-deficient Leishmaniamutant. The vaccine of the invention may optionally include a furthercompound having an immunogenic function such as a cytokine, for example,γ interferon.

In a preferred presentation, the vaccine can also comprise an adjuvant.Adjuvants in general comprise substances that boost the immune responseof the host in a non-specific manner. A number of different adjuvantsare known in the art. Examples of adjuvants may include Freund'sComplete adjuvant, Freund's Incomplete adjuvant, liposomes, and niosomesas described in WO 90/11092, mineral and non-mineral oil-basedwater-in-oil emulsion adjuvants, cytokines, short immunostimulatorypolynucleotide sequences, for example, in plasmid DNA containing CpGdinucleotides such as those described by Sato, Y. et al. 1996 Science273:352-4.

In addition, the vaccine may compromise one or more, suitablesurface-active compounds or emulsifiers, e.g. Span or Tween.

In a further aspect of the invention there is provided the use of acentrin gene-deficient Leishmania mutant as described herein for themanufacture of a vaccine for the prophylaxis and/or treatment ofleishmaniasis. Most preferably, the use is in dogs or humans.

In a further aspect of the invention there is provided a method oftreating animals that comprises administering thereto a vaccinecomposition comprising a centrin gene-deficient Leishmania mutant asdescribed herein to animals in need thereof. Preferably, the animals aredogs or humans. Naturally, the vaccine formulation may be formulated foradministration by oral dosage, by parental injection or otherwise.

The invention also provides a process for preparing a Leishmaniavaccine, which process comprises admixing a centrin gene-deficientLeishmania mutant as herein described with a suitable carrier oradjuvant.

The mode of administration of the vaccine of the invention may be by anysuitable route that delivers an immunoprotective amount of the parasiteof the invention to the subject. However, the vaccine is preferablyadministered parenterally via the intramuscular or deep subcutaneousroutes. Other modes of administration may also be employed, wheredesired, such as oral administration or via other parental routes, e.g.,intradermally, intranasally, or intravenously.

Generally, the vaccine will usually be presented as a pharmaceuticalformulation including a carrier or excipient, for example an injectablecarrier such as saline or pyrogenic water. The formulation may beprepared by conventional means. It will be understood, however, that thespecific dose level for any particular recipient animal will depend upona variety of factors including age, general health, and sex; the time ofadministration; the route of administration; synergistic effects withany other drugs being administered; and the degree of protection beingsought. Of course, the administration can be repeated at suitableintervals if necessary.

Targeted Disruption of the Centrin Gene Leads to Amastigote StageSpecific Attenuation of Growth in Leishmania donovani

Generation of LdCEN Null L. donovani Promastigotes

Homologous recombination was utilized to delete (knockout) LdCEN in thediploid organism L. donovani. To delete both alleles of the centrin genewe used two recombinant DNA fragments with two different antibioticresistant genes as markers: hyg gene that confers resistance tohygromycin B and neo gene that confers resistance to G418 (FIG. 1). Themarker genes were flanked at the 5′ and 3′ ends with the 5′ and 3′UTRsof centrin gene respectively for the homologous recombination.Construction of the targeting fragments is described in the legend toFIG. 1. Disruption of the two alleles was carried out step by step, byfirst using the hyg construct and then using the neo construct to obtaina double knockout. The clones obtained after each transfection werescreened for the deletion of centrin by both PCR analysis using primersspecific to centrin, hyg and neo genes and by Southern blot analysisusing labeled gene specific probes for centrin, hyg and neo (FIG. 2A).FIG. 2B, lane 9 shows complete loss of the coding region of the LdCEN indouble knockout mutants. Loss of centrin expression in the parasite wasalso confirmed by Northern blot analysis using LdCEN gene as probe andby Western blot analysis using anti-LdCen antibody. The centrindisrupted parasite (LdCEN^(−/−)) neither expresses the mRNA for centrinnor centrin protein (FIG. 2C, lane 3). LdCEN−/− promastigotes were thenpropagated and used for further characterization.

LdCEN^(−/−) Parasite does not Grow as an Axenic Amastigote

Our earlier studies had shown that transfected L. donovani expressingN-terminal deleted centrin grew slower compared to wild type cells invitro (Selvapandiyan, A. et al. 2001 J Biol Chem. 276:43253-43261). Inthe present disclosure deletion of both the alleles of centrin did notaffect the growth of null mutant promastigotes (FIG. 3A). However,differences in growth were seen between wild type and centrin nullmutant axenic amastigotes (FIG. 3B). The parasites were grown both aspromastigotes and axenic amastigotes in the absence of antibioticsexcept the single knockout (+/−) that was grown in the presence ofhygromycin B in order to avoid hyg gene's elimination by the duplicationof the single centrin allele. The promastigote as well as axenicamastigote form of the parasite with a single centrin allele knockout(+/−) showed a growth pattern similar to that of the wild type control(+/+) (FIGS. 3A and B). When the actively growing centrin null mutantpromastigotes (−/−) were transferred to the axenic amastigote medium,the culture showed a slight increase in cell number only in the first 24hours, a period required for the promastigotes to differentiate intoaxenic amastigotes. There after there was no growth in the culture andafter 96 hr incubation, a gradual decrease in cell number occurred (FIG.3B). It is interesting to note when a day 2 axenic amastigote culture ofLdCEN^(−/−) was switched to promastigote medium (with 100 folddilution), the parasites could differentiate into promastigotes, albeitafter a long lag period of 8 days. However, if axenic amastigotes ofLdCEN^(−/−) after 7 days in culture were shifted to promastigote medium,it was not possible to restore the growth.

LdCEN^(−/−) Axenic Amastigote Cells Fail to Duplicate Their Basal Bodies

Indirect immunofluorescence localization of centrin using a pan-centrinmonoclonal antibody (20H5) in both wild type and LdCEN deficientpromastigote and axenic amastigote cells is illustrated in FIG. 4. Bothwild type and LdCEN deficient promastigote cells showed conspicuouslocalization of centrin at the base of emergent flagella as one or twobright spots, in addition to diffuse staining along the flagellaraxoneme (FIG. 4A-B). In axenic amastigote cells, however, consistentlocalization of centrin at the flagellar base was seen only in wild typecells (FIG. 4C). Discrete centrin localization was largely absent inaxenic amastigote cells deficient for LdCEN (FIG. 4D).

High-resolution electron microscopy of the flagellar apparatus wasconducted in order to determine if specific alterations in basal bodiesor associated structures could be distinguished in LdCEN deficientpromastigote or axenic amastigote cells (FIG. 5A-D). Thin sectionsthrough the flagellar pocket revealed one or two basal bodies inpromastigote and axenic amastigote cells that were essentiallyindistinguishable between the wild type and LdCEN^(−/−) cells. Furtheranalysis demonstrated that multiple basal bodies (three or four) couldbe found in individual wild type promastigote and amastigote cells andin LdCEN deficient promastigote cells (FIG. 5E-G). However, despiteextensive analysis of sampled material, in no instance were more thantwo basal bodies found in LdCEN^(−/−) deficient amastigote cells (FIG.5H). Together with the immunofluorescence analysis these observationsindicate that expression of LdCen-p is required for basal bodyduplication in axenic amastigote cells, while basal body duplication inpromastigote cells can proceed in the absence of LdCen-p, presumablythrough the action or compensation by other Leishmania centrins.

LdCEN^(−/−) Axenic Amastigote Cells Accumulate at the G2/M Stage of theCell Cycle

To analyze the cause of the arrest of the growth of the axenicamastigote cells of LdCEN^(−/−), the axenic amastigote cultures at 24 hrand 48 hr time points were subjected to cell cycle analysis usingflowcytometry. At 24 hr culture, there were significantly lessLdCEN^(−/−) axenic amastigotes in the G1 phase and significantly more inthe G2/M phase compared to wild type axenic amastigotes (FIG. 6). Thisdifference became increasingly significant in cells after 48 hrs ofculture. Further, we also observed more LdCEN^(−/−) cells in S phase.

A characteristic feature of L. donovani axenic amastigotes in culture isthat the cells form huge cell aggregates (FIG. 7A). Under the lightmicroscope, LdCEN^(−/−) axenic amastigotes on day 2 showed fewer cellswith much smaller cell aggregates compared to the wild type axenicamastigotes (FIG. 7A). Many cells in this culture were larger in size.In order to look at the nuclei of these cells, 2 days old cultures weredispersed to individual cells, stained with DAPI and observed under thefluorescence microscope. All wild-type axenic amastigotes cells showed asingle nucleus with one kinetoplast (FIG. 7B upper panel). However, theLdCEN deficient axenic amastigotes showed cells with multiple nuclei(FIG. 7B lower panel). A large proportion of cells showed two nuclei andtwo kinetoplasts per cell. These cells were indeed at least twice thesize of the uninucleated cells. There were also cells with 4 nuclei and4 kinetoplasts and displaying much larger cell size (FIG. 7B). To obtaina more detailed picture of the nuclei and their relationship to theplasma membrane in these multinucleated cells, LdCEN^(−/−) axenicamastigotes were subject to electron microscopic analysis (FIG. 7C). Thelarge cells were multinucleated with more than one kinetoplasts. Thesecells were highly amoebiotic in shape as opposed to the wild typecontrol cells that are spherical (FIG. 7C; 2 and 3). Few of themultinucleated cells, probably the older ones, displayed condensednuclei, a feature of cells undergoing apoptosis.

In order to quantitate the multinuclear status of the LdCEN^(−/−) axenicamastigotes, we counted cells stained with DAPI in the fluorescentmicroscope. As DAPI stained the kinetoplasts brighter than the nuclei,the number of kinetoplasts per cell was counted with increasing time ofculture of the LdCEN^(−/−) axenic amastigote cells. The analysis showeda progressive increase of multi-kinetoplast cells with time in axenicamastigote culture for the centrin knockout parasites (FIG. 7D). Therewas a gradual decrease in the number of cells having a singlekinetoplast (FIG. 7D).

While counting the kinetoplasts, simultaneously we observed that therewas also an increase in the number of cells without kinetoplasts in theculture over time. An increased uptake of tryphan blue staining wasobserved in cells, which did not possess either kinetoplasts or nuclei.Considering such cells as dead cells, they were scored separately bypropidium iodide (PI) staining and analyzing using FACS. It is knownthat PI is taken up by only those cells whose plasma membrane integrityis compromised (Lee, N. et al. 2002 Cell Death Differ 9:53-64). Resultsshowed that there was a progressive increase in the number of dead cellsin LdCEN^(−/−) axenic amastigote culture over time (FIG. 8). The centrindeficient parasites showed 3 fold more PI positive cells than the wildtype control parasites after 144 hr culture as axenic amastigotes (FIG.8).

LdCEN^(−/−) Axenic Amastigote Cells Initiate Programmed Cell Death Afterthe Growth Arrest

Since there was no increase in the number of LdCEN^(−/−) axenicamastigotes in culture with time and cells were becoming PI positive, wewanted to determine the type of death pathway the multinucleated axenicamastigotes undergo. The axenic amastigotes of LdCEN^(−/−) or wild typecells were analyzed for caspase-like activity, which has been indicatedas a marker for the apoptotic death pathway in L. donovani (Arnoult, D.et al. 2002 Cell Death Differ 9:65-81; Lee, N. et al. 2002 Cell DeathDiffer 9:53-64). The percentage of cells showing caspase like activitywas measured by FACS analysis using fluorescent caspase substratePhiPhiLux (PPL). The results showed that there were more PPL positivecells in the LdCEN^(−/−) parasites in the first two days in culture thanin the wild type control (FIG. 9A). The level of PPL cleavage activitygradually decreased after 48 hr of incubation. Whereas, in control, theincrease in the level of PPL cleavage activity was observed only whenthe cells reached the stationary phase of growth in culture, as waspreviously observed by investigators (Lee, N. et al. 2002 Cell DeathDiffer 9:53-64). As further evidence of programmed cell death in thecentrin disrupted cells, TUNEL assay was carried out on the cells thatwere growing in culture for 24 and 36 hr to identify cells with thefragmented DNA characteristic of the nuclei of apoptotic cells (Lee, N.et al. 2002 Cell Death Differ 9:53-64). Few TUNEL positive cells wereobserved in the wild type cells (FIG. 9B). In contrast, in theLdCEN^(−/−) axenic amastigotes a much greater percentage of cells showedTUNEL positivity (FIG. 9B). FIG. 9C (image 6) shows the fluorescentmicroscopic picture of LdCEN^(−/−) axenic amastigote cells that are bothTUNEL positive and DAPI positive. Thus the increase in PPL cleavageactivity and the increase in the number of TUNEL positive cellsindicated that the LdCEN^(−/−) axenic amastigotes initiate programmedcell death after they become multinucleated and stop growing.

LdCEN^(−/−) Parasites do not Survive in Macrophages

Since the centrin deficient L. donovani do not grow as an axenicamastigote in vitro, we examined their survivability in macrophages. Tothis end, human monocytes newly differentiated in vitro into macrophagesby macrophage colony stimulating factor (M-CSF) were inoculated withstationary phase cultures of wild type and LdCEN^(−/−) promastigotes(FIG. 10). Results at 5 hr post infection (p.i.) showed that the percentof macrophages that take up the parasites was similar (>80%) inmacrophages inoculated with both types of parasites (FIG. 10A toppanel). These macrophages were subsequently examined at 24, 48, 120 and240 hr p.i. and the percent of infected macrophages was calculated. Thepercent of infected macrophages with LdCEN^(−/−) parasite decreased toas low as 12% at 240 hr. Whereas, at the same time, 46% of themacrophages were infected with the control parasites (FIG. 10A, toppanel). The macrophages, then, were scored for the parasite load (numberof parasites per 100 macrophages). Significant difference was observedbetween the wild type control and the LdCEN^(−/−) transfectants at 120and 240 hrs p.i. (FIG. 10A, middle and lower panels). At the 240 hr timepoint, the total number of amastigotes per 100 macrophages was ˜550 forthe wild type transfectants and only ˜26 for the macrophages transfectedwith the mutant parasite (FIG. 10A middle panel). The number ofparasites per infected host cell at 240 hr p.i. was 12 for wild typeparasite and 1.8 for the mutant parasites (FIG. 10A lower panel). After312 hr p.i., no mutant parasite was seen in the macrophages. Theseresults indicated that the centrin deficient parasites do not survive inthe macrophages in vitro. Further, LdCEN^(−/−) parasites that were takenup by macrophages developed into multinucleated large cells after 120 hrof culture (FIG. 10B). Wild type control cells did not becomemultinucleated in macrophages (FIG. 10B).

Reversal of Growth Inhibition by Centrin Expression in the LdCEN^(−/−)Parasites

To confirm that disruption of centrin gene expression was the specificcause of growth inhibition in LdCEN^(−/−) axenic amastigotes, episomalexpression of centrin in the knockout parasites was investigated. Therecombinant plasmid (pXG-PHLEO-LdCEN) transfected axenic amastigotesthat are adapted to grow in presence of Phleomycin (150 μg/ml) showedthe expression of recombinant centrin protein (tagged withhaemagglutinin tag) (FIG. 11A Lane 3). The rate of culture growth (FIG.11B) and the morphology of the cell as seen under the microscope of thecentrin re-expressing LdCEN^(−/−) knockout axenic amastigotes weresimilar to the wild type cells. There were no multinucleated cells insuch axenic amastigote culture. These studies demonstrated thatre-expression of transfected centrin in LdCEN^(−/−) parasites was ableto reverse the growth defect seen in LdCEN^(−/−) axenic amastigotes.

Discussion

The parasites of the order kinetoplastida, which infect humans, animalsand plants, are considered to be one of the earliest eukaryotes.Conventional cellular organelles of these parasites can exhibit extremefeatures rarely seen in other organisms (Bastin, P. et al. 2000 MicrobesInfect 2:1865-1874). The possible role of one such organelle, the basalbody apparatus in the parasite life cycle is still obscure. We recentlycloned and characterized a basal body protein ‘centrin’ in L. donovani,a causative agent of visceral leishmaniasis (Selvapandiyan, A. et al.2001 J Biol Chem. 276:43253-43261). Centrin in Leishmania is a calciumbinding protein implicated to be involved in growth/cell division of theparasite (Selvapandiyan, A. et al. 2001 J Biol Chem. 276:43253-43261) ashas been indicated in higher eukaryotes (Pastrana-Rios, B. et al. 2002Biochemistry 41:6911-6919; Salisbury, J. L. et al. 2002 Curr Biol12:1287-1292; Wiech, H. et al. 1996 J Biol Chem 271:22453-22461). Todefine the decisive role of centrin in this parasite, we aimed atdisrupting the centrin gene in L. donovani. Since Leishmania is diploidthroughout in its life cycle, we adopted deletion of the centrin geneusing two antibiotic resistant targeting genes. One potential problemfor gene targeting for Trypanosomatid genes is, that, most of theproteins are encoded by arrays of multiple genes (Cruz, A. et al. 1991PNAS USA 88:7170-7174). Since our earlier work had indicated that LdCENis a single copy gene in the Leishmania genome (Selvapandiyan, A. et al.2001 J Biol Chem. 276:43253-43261), we could disrupt the gene by thedeletion of its alleles with hyg and neo constructs. The expression ofcentrin in L. donovani correlates with active growth of bothpromastigotes and axenic amastigotes in vitro (Selvapandiyan, A. et al.2001 J Biol Chem. 276:43253-43261). Therefore we wanted to explore theeffect of LdCEN deletion on the growth of the parasite. Parasitesdisrupted for a single LdCEN allele did not show any significantdifference in their growth compared to control. The growth of thecentrin double allele null mutant parasite, as promastigote was notaffected in vitro. However, the growth of the axenic amastigote wassignificantly affected both in vitro as well as in the humanmacrophages. Upon re-expressing centrin in the mutant parasite throughan episome, the growth inhibition in the axenic amastigote stage wasabolished and the cells resumed normal growth. These experiments extendthe importance of this gene for the growth of the parasite, mainly forthe amastigote form.

Analysis by indirect immunofluorescence demonstrated centrinlocalization at the flagellar base in wild type and LdCEN^(−/−)promastigote cells and in wild type amastigote cells. Axenic amastigotecells lacking the LdCEN gene and LdCen-p expression showed greatlyreduced centrin fluorescence in most cells. Electron microscopy showedthat flagellar apparatus and basal body structure was essentiallyindistinguishable between wild type and LdCEN^(−/−) cells, albeit basalbody duplication was evident only in promastigote and wild type axenicamastigote cells. These observations identify centrin in the normalduplication process of amastigote basal bodies and indicate that centrinfunction is either substituted or compensated for by other Leishmaniacentrins in the LdCEN^(−/−) promastigote stage cells.

We further analyzed the cause of the growth defect through cell cycleanalysis of the LdCEN^(−/−) axenic amastigote. The flowcytometryanalysis on LdCEN^(−/−) axenic amastigote culture showed a significantlygreater number of cells in the G2/M cell cycle stage. Both light andelectron microscopy revealed that beyond 48 hr of incubation, many ofthese cells were large, without definite shape and with either two ormore nuclei and with as many kinetoplasts. The multinucleated natureindicates the lack of cytokinesis in these cells. Gene silencing forHsCEN2 via RNAi in cultured HeLa cells results in failure of centrioleduplication during the cell cycle and the cells give rise tomultinucleate cells and finally die (Salisbury, J. L. et al. 2002 CurrBiol. 12: 1287-1292). A similar result also was noticed upon silencingthe centrin gene by RNAi in Chlamydomonas (Koblenz, B. et al. 2003 JCell Sci 116:2635-2646). The interference in the cytokinesis in centrinRNAi cells of Chlamydomonas was due to the presence of aberrant numbersof basal bodies that were separated from the spindle poles. Severalmechanisms can explain failed cell division. Basal body/centrioleduplication occurs once per cell division, and failed duplication orover production of these organelles would lead to aberrant cell divisionphases (Koblenz, B. et al. 2003 J Cell Sci 116:2635-2646; Salisbury, J.L. 2003 Curr Biol 13:R88-90; Salisbury, J. L. et al. 2002 Curr Biol,12:1287-1292). These results demonstrate a requirement for centrin inthe cytokinesis during mitosis and also shed some light on thedifferences in the molecular mechanism of cell division between the twostages of the parasite. The occurrence of a progressive increase in thenumber of multinucleated cells, closely followed by an increase in thenumber of dead cells indicates that the axenic amastigotes of theLdCEN^(−/−) parasite die after going through a few rounds of nucleardivisions without cytokinesis. Basal bodies and centrioles share thesame highly conserved ultrastructure of nine triplet microtubulesforming a cylinder (Lechtreck, K. F. and Bornens, M. 2001 Eur J CellBiol 80:631-641). The requirement of Human centrin 2 in centrioleduplication, as well as a role in organizing spindle pole morphology andin the completion of cytokinesis was previously reported (Salisbury, J.L. et al. 2002 Curr Biol. 12:1287-1292). Similarly, by the presentdisclosure we observed the requirement of centrin for the completion ofcytokinesis in Leishmania. The mechanism by which centrin involvement inthe basal body duplication and in organizing other cytoskeletalmorphology needs to be studied in this evolutionarily primitive organism(L. donovani), using centrin null mutant parasites.

Since we observed no growth in multinucleated LdCEN^(−/−) axenicamastigotes and an increased number of cells permeable to PI, anindication of cell death in such cells, we analyzed the mode of celldeath in such cells. Previously investigators have shown that cellgrowth arrest at saturation densities in culture leads to programmedcell death in Leishmania (Lee, N. et al. 2002 Cell Death Differ9:53-64). In the present disclosure, we observed an increased PPLcleavage activity and TUNEL positivity in centrin deficient axenicamastigotes, which indicates that these cells are initiating theapoptotic pathway once cell division is disrupted.

The differential effect of centrin gene knockout in amastigotes isunique and surprising. We have evidence that LdCen-p is encoded by asingle copy gene (Selvapandiyan, A. et al. 2001 J Biol Chem.276:43253-43261). In addition, the level of expression of LdCen proteinwas similar in both promastigotes and axenic amastigotes (Selvapandiyan,A. et al. 2001 J Biol Chem. 276:43253-43261). However, we also hadobserved the existence of more than one centrin type in L. donovani(Selvapandiyan, A. et al. 2001 J Biol Chem. 276:43253-43261). Recently,we observed additional centrin related genes in Leishmania major in thegenome data bank. Therefore it is possible that different centrin genes,which may have a stage specific differential expression, couldcomplement centrin function only for promastigotes in the LdCENdisrupted parasite. Alternatively the existence of amastigote specificgrowth regulating protein(s) that require interaction with centrin,could fail to function in the LdCEN^(−/−) amastigotes. In our earlierreport describing the dominant negative effect in the slow growingparasites expressing N-terminal deleted centrin, we had speculated aboutprotein interaction involving L. donovani centrin and some yet unknownproteins (Selvapandiyan, A. et al. 2001 J Biol Chem. 276:43253-43261).In yeast, proteins KAR1 and CDC31 (yeast centrin) are required for theinitial stages of spindle pole body duplication (Vallen, E. A. et al.1994 Genetics 137:407-422). By immunofluorescence microscopy theinteraction between these two proteins have been demonstrated in vivo(Biggins, S. and Rose, M. D. 1994 J Cell Biol 125:843-852). However, incontrast to the stage specific growth inhibition observed with theLdCEN^(−/−) Leishmania (present disclosure), both promastigote and theamastigote stages were equally affected when N-terminal deleted centrinwas expressed in the parasite (Selvapandiyan, A. et al. 2001 J BiolChem. 276:43253-43261). Discovery of a novel centrin-binding partner,Sfi1p, lends new insight into the functional properties of centrin(Kilmartin, J. V. 2003 J Cell Biol 162:1211-1221; Salisbury, J. L. 2004Curr Biol 14:R27-29). Sfi1 protein binds multiple centrin moleculesalong a series of internal repeats, and the Sfi1p/centrin complex formsCa²⁺-sensitive contractile fibers that function to orient or positioncentrioles/basal bodies and to alter centrosome structure. Geneticanalysis in yeast and Chlamydomonas and centrin ablation by siRNA orgene knockout clearly demonstrates that centriole/basal body duplicationdepends on proper Sfi1p/centrin function (see Salisbury, J. L. 2004 CurrBiol 14:R27-29, for review). Taken together these results predict a verycomplex mode of function and regulation of centrin in thisevolutionarily primitive parasite, Leishmania. The present disclosure,however, demonstrates that LdCen protein is essential for the parasiteto survive, especially for the amastigote stage.

Similar studies of gene knockouts for various enzyme cell surface andtransporter proteins and the effect on virulence of the null mutantLeishmania parasites have been carried out by different laboratories.Examples are: dihydrofolate reductase-thymidylate synthase (Cruz, A. etal. 1991 PNAS USA 88:7170-7174; Titus, R. G. et al. 1995 PNAS USA92:10267-10271), pteridine reductase 1 (Bello, A. R. et al. 1994 PNASUSA 91:11442-11446; Cunningham, M. L. et al. 2001 Science 292:285-287),lipophosphoglycan (Spath, G. F. et al. 2000 PNAS USA 97:9258-9263;Spath, G. F. et al. 2003 PNAS USA 100:9536-41) and leishmanolysin genes(Joshi, P. B. et al. 2002 Mol Biochem Parasitol 120:33-40) in L. major,and the glucose transporter gene family (Burchmore, R. J. et al. 2003PNAS USA 100:3901-3906) in L. mexicana and ornithine decarboxylase(Jiang, Y. et al. 1999 J Biol. Chem. 274:3781-3788), and spermidinesynthase (Roberts, S. C. et al. 2001 Mol Biochem Parasitol 115:217-226)in L. donovani. In all the above studies, the gene knockout has affectedequally both the promastigote and amastigote stages of the parasite.However, interestingly, in this disclosure we observed a differentialeffect that L. donovani centrin gene disruption affects selectively onlythe growth of the amastigote stage of the parasite both in vitro andinside the human macrophages. This is the first report that describes agene knockout for a cytoskeletal structural protein in Leishmania andthe importance of such a gene for the growth of the parasite. Thepractice of gene knockout realized for centrin in L. donovani can beapplied readily for other Leishmania sp. that have a high degree ofgenome sequence conservation and cause similar visceral leishmaniasis,e.g., L. infantum and L. chagasi. In addition, inactivation of thecentrin gene in the amastigote form of other related Leishmania species(L. major, L. braziliensis), which cause other forms of leishmaniasis,such as cutaneous and mucocutaneous diseases, is obvious as beingutilized in the development of attenuated strains for these otherspecies of Leishmania. Such attenuated strains are envisioned as beingprepared as vaccines for these diseases.

Example I

In Vitro Culture of Parasites

The cloned line of L. donovani designated by the World HealthOrganization as MHOM/SD/62/1S-C1_(2D) (SD) (Joshi, M. et al. 1995 JEukaryot Microbiol 42:628-632) was used in all the experiments.Promastigotes and the axenic amastigotes were grown and harvested asdescribed previously (Joshi, M. et al. 1993 Mol Biochem Parasitol58:345-354). Wherever needed the parasites were counted visually underthe microscope (ECLIPSE TE2000-U; Nikon corporation, Tokyo, Japan). Inthe experiments Student's t-test was used to determine the significance.

Construction of DNA for Targeted Gene Deletion

For L. donovani centrin gene knockout, nearly two thirds of the openreading frame (orf) from the 5′ end (323/447 bp) was replaced with theorf of the selectable marker gene comprising either the hyg gene or neogene. These genes were flanked at the 5′ end by the 1198 bp upstreamregion of the LdCEN gene and at the 3′ end by the 124 bp 3′ region ofthe LdCEN orf followed by 716 bp of its downstream region. (A) The 5′UTRof LdCEN was amplified by PCR using a cosmid clone containing the LdCENgene as template (Selvapandiyan, A. et al. 2001 J Biol. Chem.276:43253-43261) and the following forward (P1) and reverse (P2)primers. P1: 5′-GGG ATC CTT ATA GCC ACG GAT G-3′ (SEQ ID NO: 7) containsa BamHI restriction site (bold). P2: 5′-GGG TCG ACC CAC AAA AAG AAATTG-3′ (SEQ ID NO: 8) contains a SalI restriction site (bold). Theresulting PCR product was cloned directly at the T/A cloning site ofpCRII-TOPO cloning vector (Invitrogen Co.). The recombinant plasmidhaving the insert with the SalI end towards Sp6 promoter was cut withSpeI and KpnI and used in the next step. (B) The 3′UTR of LdCEN was PCRamplified using the cosmid clone mentioned above as template and thefollowing forward (P3) and reverse (P4) primers. P3: 5′-GAC TAG TAC TGCTGG GTG AGA ACC-3′ (SEQ ID NO: 9) contains a SpeI restriction site(bold). P4: 5′-GGG TAC CTA TTT ATC GCC TGC TCG G-3′ (SEQ ID NO: 10)contains a KpnI restriction site (bold). The PCR product was restrictedwith SalI and KpnI and ligated at the same sites of the cut plasmidproduct of step A. The resulting recombinant plasmid was cut with SalIand SpeI and used in step C. (C) The selectable marker sequence hyg wasamplified using plasmid pX63-HYG (Cruz, A. et al. 1991 PNAS USA88:7170-7174) as template and forward (P5) and reverse (P6) primers. Theneo gene was amplified using plasmid pKSNEO (Zhang, W. W. et al. 1996Mol Biochem Parasitol 78:79-90) as template and forward (P7) and reverse(P8) primers. To ensure translation of these marker genes, nucleotides−16-+12 of 3′-nucleotidase/nuclease (3′NT/Nu) gene including thetranslation initiation site ‘ATG’ (Debrabant, A. et al. 2000 J Biol Chem275:16366-16372; Debrabant, A. et al. 1995 Mol Biochem Parasitol71:51-63) was added upstream of hyg and neo genes (M-seq; FIG. 1) duringPCR amplification. This was based on our earlier experience ofsuccessful gene targeting to eliminate 3′NT/Nu gene. P5: 5′-G GTC GACGCT ACG GCA GAC ATG GCT CGA GCT CTG atg aaa aag cct gaa ctc-3′ (SEQ IDNO: 11) contains sequentially a SalI restriction site (bold), 3′NT/Nusequence as mentioned earlier (single underlined) and 18 nucleotide hygsequence from beginning of orf (lower case). P6: 5′-GGA CTA GTC TAT TCCTTT GCC CTC G-3′ (SEQ ID NO: 12) has a SpeI restriction site (bold)followed by 3′ end sequence of hyg gene. P7: 5′-G GTC GAC GCT ACG GCAGAC ATG GCT CGA GCT CTG atg att gaa caa gat gga-3′ (SEQ ID NO: 13) issimilar to primer P5 except that at its end instead of hyg sequence, ithad 18 nucleotides neo sequence from beginning of orf (lower case). P8:5′-GAC TAG TCA GAA GAA CTC GTC AAG-3′ (SEQ ID NO: 14) has a SpeIrestriction site (bold) followed by 3′ end sequence of neo gene. Boththe PCR amplified hyg and neo fragments were cut with SalI and SpeIrestriction enzymes and the fragments were ligated at the cut plasmidobtained at step B. The authenticity of each of the constructs and thefidelity of the PCR amplified fragments were verified by nucleotidesequencing. The resulted hyg and neo recombinant plasmids were cut withBamHI and KpnI and the fragments of either hyg or neo flanked with theUTR sequences of LdCEN were individually used for transfection todisrupt LdCEN gene in L. donovani.

Transfection and Selection of LdCEN Null Mutant

Mid-log phase promastigotes (2−4×10⁷ cells/ml) were harvested bycentrifugation at 3000 g for 10 min at 4° C. Cell pellets were washed inice-cold PBS and electroporated with the DNA using conditions asdescribed previously (Selvapandiyan, A. et al. 2001 J Biol Chem.276:43253-43261). For clonal selection in general, the transfectedpromastigotes were incubated overnight in 5 ml Leishmania growth medium(Joshi, M. et al. 1995 J Eukaryot Microbiol 42:628-632). The followingday the pellet of the culture was resuspended in 200 μl of thepromastigote growth medium, syringe disrupted with 28G needle and platedon 1% agar plate containing the same growth medium supplemented with 4μg/ml Biopterin (Calbiochem) from stock 200 μg/ml in 10 mM NaOH andrespective antibiotic/s. The antibiotic unless otherwise mentioned G418(Geneticin; BRL) was present at 40 μg/ml in plates and 20 μg/ml inliquid culture and corresponding concentrations of hygromycin B (Sigma)were 80 and 40 μg/ml respectively.

Transfection with construct #1 (FIG. 1) was performed as described aboveand finally plated on semi-solid plate containing hygromycin B. Clonesisolated from the plates were subsequently expanded in liquid mediumcontaining hygromycin B. Genomic DNA isolated from these clonedparasites was used in PCR and Southern blot analyses to confirm the lossof one allele of centrin. Such a cell line in which one allele of thecentrin gene was substituted by the hyg gene was subjected to the nextround of transfection using Construct #2 and clones were selected on theplate in the presence of both hygromycin B and G418 antibiotics. Theclones were analyzed for the complete knockout of centrin, and thepresence of both hyg and neo genes using PCR, Southern and Western blotanalyses. The knockout clone was then propagated and used in subsequentexperiments.

Southern and Western Blot Analyses

Isolation and Southern analysis of genomic DNA from L. donovani wereperformed as described previously (Selvapandiyan, A. et al. 2001 J BiolChem. 276:43253-43261). Extraction of protein from the parasite andanalysis by Western blot were also conducted as described previously(Selvapandiyan, A. et al. 2001 J Biol Chem. 276:43253-43261).

Light and 4′6-diamidino-2-phenylindole (DAPI)-Fluorescence Microscopy

For DAPI-fluorescence the parasites grown at different time points werewashed once with 1×PBS and treated with tryphan blue. The cells werethen air dried on the microscopic slide mounted in Vectashieldcontaining 4′6-diamidino-2-phenylindole (DAPI)(Vector Laboratories,Inc.) to stain both nucleus and kinetoplast. Cells were examined forfluorescence under the ECLIPSE TE2000-U microscope (Nikon corporation,Tokyo, Japan) with epifluorescence and images captured with a digitalcamera (C4742-95), Hamamatsu Photonics K.K., Japan and processed withOpen Lab software (Improvision, Inc.). Densely fluorescing kinetoplasts,observed in the viable cells (cells non stained with tryphan blue) werecounted visually. For indirect immunofluorescence studies, 200 μl ofcultured cells were harvested and mounted on slides using a Cytospin3(Shandon, Pittsburgh Pa.). The preparation was fixed in −20° C. methanolfor 10 min, blocked (10% goat serum, 5% glycerol, 0.1% NP40 in PBS) for30 min and incubated with 1:2000 anti-centrin (20H5 ascites fluid) for30 min. The preparation was then washed thoroughly with PBS, incubatedin FITC conjugated secondary antibody for 30 min, washed again andstained for DNA using DAPI.

Electron Microscopy

Fixing of axenic amastigote cultures of wild type (+/+) and centrindeficient (−/−) parasites from 48 hr incubation point, embedding inepoxy resin, sectioning, staining and examining with an electronmicroscope were performed as described previously (Lee, N. et al. 2002Cell Death Differ 9:53-64).

Flowcytometry

Axenic amastigotes from 24 and 48 hr cultures were collected, fixed in70% ethanol, stained with 50 μg/ml propidium iodide (PI from Sigma) inPBS and analyzed as described previously (Selvapandiyan, A. et al. 2001J Biol Chem. 276:43253-43261). The data were analyzed using the ModfitLt. Software, Verity Software House, Inc. (Topshan, Me.). For eachsample 20,000 fluorescent events were measured. To analyze the plasmamembrane integrity and PhiPhiLux (PPL) cleavage activity, promastigoteswere inoculated at 1×10⁶ cells/ml into axenic amastigote culture medium(Joshi, M. et al. 1993 Mol Biochem Parasitol 58:345-354) and samples ofaxenic amastigote cells collected at various times after inoculation.Fluorescence of the cells treated with both PI and PPL (Calbiochem, LaJolla, Calif., USA) was measured following published protocols (Lee, N.et al. 2002 Cell Death Differ 9:53-64). The data are expressed aspercent of either PI or PPL positive parasites. All the above analyseswere carried out using FACScan flow cytometer (Becton DickinsonImmunocytometry Systems, San Jose, Calif.) and CELLQuest software. Foreach sample 10,000 fluorescent events were measured.

TUNEL Assay

LdCEN^(−/−) and wild type promastigotes were inoculated into axenicamastigote culture medium and allowed to grow for different periods oftime, washed with PBS, fixed in 4% para-formaldehyde for 30 min, rinsedwith PBS, permeabilized with 0.1% Triton X-100 in 0.1% sodium citratefor 2 min, rinsed with PBS and incubated with 50 μl per sample of TUNELreaction mixture (Roche Diagnostics GmbH), containing FITC-labeled dUTPand terminal deoxynucleotidyl transferase, for 1 hr at 37° C. A negativecontrol was included in each staining wherein only the labeling solutionwas added. To assess the nuclear morphology of the cells, the sampleswere stained, by mounting with Vectashield (Vector Laboratories, Inc.)containing DAPI. The specimens were then observed under a fluorescencemicroscope. DAPI fluorescent images were captured through a 364 nmwavelength filter and pseudocolored red to enhanced the contrast withthe FITC image.

In Vitro Macrophage Infections

Human elutriated monocytes were resuspended at 1.8×10⁵ cells/ml in RPMImedium containing macrophage colony stimulating factor (Debrabant, A. etal. 2002 Int J Parasitol 32:1423-1434), plated in 0.5 ml on eightchamber Lab-Tek tissue culture slides (Miles Laboratories) and incubatedfor 8 days for differentiation into macrophages. The differentiatedmacrophages were infected with stationary phase cultures ofpromastigotes (10:1, parasite to macrophage ratio) as describedpreviously (Debrabant, A. et al. 2002 Int J Parasitol 32:1423-1434).After incubation for 5 hr at 37° C. in 5% CO₂, the free extracellularparasites were removed by repeated washings in RPMI, and the cultureswere incubated in macrophage culture medium for maximum 240 h. At 5, 24,48, 120 and 240 hr post infection (p.i.), the culture medium was removedfrom a sample of the culture slides, the slides were air-dried, fixed byimmersion in absolute methanol for 5 min at room temperature and stainedusing Diff-Quick Stain set (Baxter Healthcare Corporation, Miami, Fla.).For each culture, a minimum of 300 macrophages were counted. Values areexpressed either as percentage of macrophages that were infected byLeishmania, as the total number of amastigotes per 100 macrophage cellsobserved (infected plus noninfected) or as the mean number of parasitesper infected macrophage.

Restoration of Centrin in LdCEN^(−/−) Parasites

In order to restore centrin in the LdCEN^(−/−) parasites, LdCEN orf wasfirst PCR amplified using a LdCEN containing plasmid (Selvapandiyan, A.et al. 2001 J Biol Chem. 276:43253-43261) as template and the followingforward (P9) and reverse (P10) primers. P9: 5′-GGG ATC CAT GGC TGC GCTGAC GGA T-3′ (SEQ ID NO: 15) contains a BamHI restriction site (bold).P10: 5′-GGG ATC CCT ACG CGT AGT CCG GCA CGT CGT ACG GGT Act ttc cac gcatgt gca g-3′ (SEQ ID NO: 16) contains sequentially a BamHI restrictionsite, a sequence for an haemagglutinin tag (underlined) and 18nucleotides of 3′-end of the LdCEN gene sequence (lower case). Theamplified product was initially cloned at the T/A cloning site ofpCRII-TOPO cloning vector (Invitrogen Co.). The fidelity of the clonedsequence was verified by nucleotide sequencing. The BamHI insert wassubsequently cloned at the same cite of pXG-PHLEO vector (Engel, M. L. Iet al. 2001 Nucleic Acids Res 29:725-731) and the recombinant plasmid,pXG-PHLEO-LdCEN, was transfected into the LdCEN^(−/−) promastigotes asdescribed previously (Selvapandiyan, A. et al. 2001 J Biol Chem.276:43253-43261). Transfected promastigotes were selected with minimaldoses of Phleomycin (Sigma) (10 μg/ml). In our experience, expression ofprotein through an episome is enhanced by increasing the concentrationof selection drug in the medium. Hence, the selected promastigotes aftereach culture cycle were grown in gradually increasing Phleomycin levelsup to 150 μg/ml. Parasites grown in the presence of 150 μg/ml of drugwere used in subsequent experiments.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

What is claimed is:
 1. An attenuated strain of Leishmania from the groupconsisting of Leishmania donovani, Leishmania infantum, Leishmaniachagasi, Leishmania major, Leishmania tropica, Leishmania aethiopica,Leishmania braziliensis, Leishmania mexicana, and Leishmaniaamazonensis, wherein both wild-type copies of the single copy geneencoding centrin have been deleted to provide a strain incapable ofexpressing native centrin protein and having a reduced ability to infector survive in macrophages in comparison to wild-type strain.
 2. Thestrain of claim 1, wherein said deletion of at least one wild-type copyof the single copy gene leaves a gap into which is inserted a firstmarker gene.
 3. The strain of claim 1 wherein said first marker geneencodes antibiotic resistance.
 4. The strain of claim 3, in which saidfirst marker gene encodes antibiotic resistance to a first antibiotic,and wherein said deletion of the other wild-type copy of the single copygene leaves a gap into which is inserted a second marker gene, andwherein said second marker gene encodes antibiotic resistance to asecond antibiotic.
 5. An immunogenic composition comprising theattenuated strain of claim
 1. 6. A method of generating an immuneresponse in a host comprising administering thereto an immunoeffectiveamount of the immunogenic composition of claim 5.